| Project/Area Number |
15590945
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Metabolomics
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| Research Institution | EHIME UNIVERSITY |
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Principal Investigator |
HASHIRAMOTO Mitsuru EHIME UNIVERSITY, University Hospital, instructor, 医学部附属病院, 助手 (40346680)
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| Co-Investigator(Kenkyū-buntansha) |
MAKINO Hideichi EHIME UNIVERSITY, School of medicine, Professor, 医学部, 教授 (50009578)
OSAWA Haruhiko EHIME UNIVERSITY, School of medicine, Associate professor, 医学部, 助教授 (90294800)
ONUMA Hiroshi EHIME UNIVERSITY, School of medicine, instructor, 医学部, 助手 (00294794)
NISHIDA Wataru EHIME UNIVERSITY, University hospital, instructor, 医学部附属病院, 助手 (80271089)
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| Project Period (FY) |
2003 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| Budget Amount *help |
¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 2004: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2003: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Keywords | insulin action / glucose transport / glucose transporter / GLUT4 / retrovirus expression system |
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| Research Abstract |
The initial experimental plan was (1)to express exofacial myc-tagged GLUT4 cDNA into "insulin-stimulated GLUT4 translocation-incompetent" cells, (2)to infect cDNA library generated from "insulin-stimulated GLUT4 translocation-competent" 3T3-Ll adipocytes by using retrovirus expression system, and (3)to screen infected monoclonal clones by flow cytometry analysis (FACScan) with their immunofluorescent labelling of the exofacial myc epitope, thereby (4)isolating the clones, which show increase in the exofacial fluorescent labeling, indicative of insulin-stimulated translocation of GLUT4 to the cell surface. These were aimed to directly identify gene(s), involved in the "insulin-stimulated GLUT4 translocation" process and were based on the Fluorescence Localization-based Retrovirus-mediated Expression cloning (FL-REX) method, previously established and reported by Kitamura et al. (Proc Natl Acad Sci USA. 97: 3062, 2000). After the identification of the gene(s), we planned (5)to do PCR analysis to reach the identity of the gene(s) and eventually (6)to investigate the functional role of the isolated gene(s) in insulin-stimulated GLUT4 translocation process by using various molecular and cellular biological techniques.
We have already finished most of the screening procedures and succeeded in isolating multiple clones, which showed positive results in the preceding steps [(1)-(4) of the above]. Based on these results, we are currently doing the subsequent step (5) and will soon investigate the functional aspects of these genes by cellular biological methods (6).
We will also keep on tracking the screening steps to further isolate positive clones, by using an alternative screening method, developed newly during the course of the above experiments.
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